Peptides having osteoblast growth-promoting activity and use thereof

ABSTRACT

The present invention provides a novel peptide having bone formation-promoting effect and chondrocyte growth-promoting effect, in particular, a peptide having osteoblast growth-promoting activity, having 100 amino acid residues or less comprising an amino acid sequence selected from 
     
       
         
               
             
                 (a) 
               
                 (SEQ ID NO: 1) 
               
                 Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser- 
               
                 Pro-Tyr-Glu, 
               
           
              
              
              
              
             
          
         
       
         
         
           
             (b) an amino acid sequence derived from the amino acid sequence (a) by conservative substitution or deletion of 1 to 3 amino acids, and 
             (c) an amino acid sequence consisting of at least four contiguous amino acids of the amino acid sequence (a) or (b), or 
             a derivative thereof or a salt thereof.

TECHNICAL FIELD

The present invention relates to a peptide having osteoblastgrowth-promoting activity; a bone formation promoter, a bone resorptioninhibitor, a chondrocyte growth promoter, a hyaluronic acid productionpromoter, a chondrogenic cell differentiation inducer, a mesenchymalstem cell growth promoter and a mesenchymal stem cell differentiationinducer, each comprising the peptide; and use thereof.

BACKGROUND ART

Osteoporosis is a systemic disease that develops due to reduction of thebone volume or the bone mineral content and causes the breakage of thefine structure of bone, which reduces the bone strength and increasesthe risk of bone fracture. The number of patients with osteoporosis inJapan reaches about 11 million, and 80% of them are women. Osteoporosismainly affects people in their middle or older age, and considering thatthe society is aging, the number of patients is expected to increase inthe future. Another concern is that the incidence of bone fracture isincreasing in elementary and junior high school students. In order toprevent osteoporosis, it is very important to substantially increase thebone mineral content during the young age, thus achieving a high peakbone mass. Thus the maintenance of bone health is now a social interestregardless of sex and age.

Bone diseases are conventionally prevented or treated by dietary calciumsupplementation, light exercise, sun bath, medication, etc. Such dietarycalcium supplementation is done with calcium salts such as calciumcarbonate and calcium phosphate, and natural calcium sources such asbovine bone powder, egg shells and fish bone powder. However, suchingredients are not very suitable for oral ingestion in terms ofsolubility, absorptivity and taste. Moderate exercise increases the bonevolume and strengthens bone, and a stroll and a walk are good for bonehealth. However, for people with low physical strength, even lightexercise is troublesome, and exercise is almost impossible for thebedridden elderly. Sun bath is considered to be good for supply ofactivated vitamin D₃, but sun bath is insufficient for prevention ortreatment of bone diseases.

For the prevention or treatment of various chondropathies, the growth ofchondrocytes and the expression of differentiation function areimportant. That is, the growth and maturation of chondrocytes areconsidered to promote normal growth of bone and repair bone fracture.Several factors for inducing the growth of chondrocytes have beenreported, including transforming growth factor (TGF)-β1, insulin-likegrowth factor (IGF)-1, basic fibroblast growth factor (bFGF),PTH-related peptide (PTHrP), hepatocyte growth factor (HGF), and bonemorphogenetic protein (BMP). However, clinical applications ofchondrocyte growth-promoting drugs excellent in safety, stability andefficacy have not been established yet.

Patients with osteoarthritis account for the largest proportion ofchondropathy patients. One of the causes of osteoarthritis may be aging,and the incidence of the disease is expected to increase in this agingsociety. Conventionally, bone resorption inhibitors such as estrogen andcalcitonin, aspirin, and nonsteroidal anti-inflammatory drugs (NSAIDs)have mostly been used for the prevention and treatment of cartilagedisorders that manifest degeneration of cartilage as major symptoms,such as joint diseases. However, these drugs are not effective enough,and are well-known to cause adverse reactions such as digestive tractdisorders. Under these circumstances, there has been a great demand forprophylactic or alleviating drugs that are safe for use in the treatmentof cartilage injuries and cartilage disorders.

The inventors found that an egg yolk-derived peptide comprising an aminoacid sequence comprising at least Ala-Glu-Ser has osteoblastgrowth-promoting activity (see Patent Literature 1) and that an egg-yolkprotein hydrolysate has chondrocyte growth-promoting activity (seePatent Literature 2). The inventors then filed a patent based on thesefindings.

CITATION LIST Patent Literature

Patent Literature 1: JP 2011-211979 A

Patent Literature 2: WO 2014/007318

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a novel peptide havingbone formation-promoting effect and chondrocyte growth-promoting effectand to provide excellent applications of the peptide.

Solution to Problem

The present invention has been made to solve the above problems andincludes the following.

[1] A peptide having osteoblast growth-promoting activity, having 100amino acid residues or less comprising an amino acid sequence selectedfrom

(a) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu (SEQ IDNO: 1),

(b) an amino acid sequence derived from the amino acid sequence (a) byconservative substitution or deletion of 1 to 3 amino acids, and

(c) an amino acid sequence consisting of at least four contiguous aminoacids of the amino acid sequence (a) or (b), or

a derivative thereof or a salt thereof.

[2] The peptide of the above [1] or a derivative thereof or a saltthereof, which has at least one phosphorylated serine.

[3] The peptide of the above [1] or [2] or a derivative thereof or asalt thereof, which is a fragment of lipovitellin-1.

[4] The peptide of any one of the above [1] to [3] or a derivativethereof or a salt thereof, which further has chondrocytegrowth-promoting activity and/or hyaluronic acid production-promotingactivity.

[5] The peptide of any one of claims 1 to 4 or a derivative thereof or asalt thereof, which consists of an amino acid sequence selected from

(i) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu, (ii) (SEQ ID NO: 2) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu, (iii)(SEQ ID NO: 3) Val-Asn-Pro-Glu-Ser-Glu-Glu, (iv) (SEQ ID NO: 4)Pro-Glu-Ser-Glu-Glu, (v) (SEQ ID NO: 5) Asp-Glu-Ser-Ser-Pro-Tyr-Glu, and(vi) (SEQ ID NO: 6) Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu.

[6] A polynucleotide encoding the peptide of any one of the above [1] to[5].

[7] An expression vector comprising the polynucleotide of the above [6].

[8] A transformant resulting from transformation with the recombinantvector of the above [7].

[9] A bone formation promoter comprising the peptide of any one of theabove [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[10] The bone formation promoter of the above [9], which has osteoblastgrowth-promoting effect.

[11] A bone resorption inhibitor comprising the peptide of any one ofthe above [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[12] A chondrocyte growth promoter comprising the peptide of any one ofthe above [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[13] A hyaluronic acid production promoter comprising the peptide of anyone of the above [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[14] A chondrogenic cell differentiation inducer comprising the peptideof any one of the above [1] to [5] or a derivative thereof or a saltthereof or lipovitellin-1 or a derivative thereof or a salt thereof.

[15] A mesenchymal stem cell growth promoter comprising the peptide ofany one of the above [1] to [5] or a derivative thereof or a saltthereof or lipovitellin-1 or a derivative thereof or a salt thereof.

[16] A mesenchymal stem cell differentiation inducer comprising thepeptide of any one of the above [1] to [5] or a derivative thereof or asalt thereof or lipovitellin-1 or a derivative thereof or a saltthereof.

[17] The promoter, inhibitor or inducer of any one of the above [9] to[16], which is for oral administration.

[18] A medicament comprising the peptide of any one of the above [1] to[5] or a derivative thereof or a salt thereof or lipovitellin-1 or aderivative thereof or a salt thereof.

[19] The medicament of the above [18], for promoting bone formation orfor preventing or alleviating a cartilage disorder or a joint disease.

[20] A food or drink product comprising the peptide of any one of theabove [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[21] The food or drink product of the above [20], for promoting boneformation or for preventing or alleviating a cartilage disorder or ajoint disease.

[22] A supplement comprising the peptide of any one of the above [1] to[5] or a derivative thereof or a salt thereof or lipovitellin-1 or aderivative thereof or a salt thereof.

[23] The supplement of the above [22], for promoting bone formation orfor preventing or alleviating a cartilage disorder or a joint disease.

[24] A food additive comprising the peptide of any one of the above [1]to [5] or a derivative thereof or a salt thereof or lipovitellin-1 or aderivative thereof or a salt thereof.

[25] The food additive of the above [24], for promoting bone formationor for preventing or alleviating a cartilage disorder or a jointdisease.

[26] A cosmetic product comprising the peptide of any one of the above[1] to [5] or a derivative thereof or a salt thereof or lipovitellin-1or a derivative thereof or a salt thereof.

[27] The cosmetic product of the above [26], for promoting boneformation or for preventing or alleviating a cartilage disorder or ajoint disease.

[28] A method for promoting bone formation, the method comprisingadministering, to a mammal, an effective amount of the peptide of anyone of the above [1] to [5] or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

[29] A method for preventing or alleviating a cartilage disorder or ajoint disease, the method comprising administering, to a mammal, aneffective amount of the peptide of any one of the above [1] to [5] or aderivative thereof or a salt thereof or lipovitellin-1 or a derivativethereof or a salt thereof.

[30] The peptide of any one of the above [1] to [5] or a derivativethereof or a salt thereof or lipovitellin-1 or a derivative thereof or asalt thereof for use in the promotion of bone formation or theprevention or alleviation of a cartilage disorder or a joint disease.

[31] Use of the peptide of any one of the above [1] to [5] or aderivative thereof or a salt thereof or lipovitellin-1 or a derivativethereof or a salt thereof for the promotion of bone formation or for theprevention or alleviation of a cartilage disorder or a joint disease.

[32] Use of the peptide of any one of the above [1] to [5] or aderivative thereof or a salt thereof or lipovitellin-1 or a derivativethereof or a salt thereof for the production of a medicament forpromoting bone formation or for preventing or alleviating a cartilagedisorder or a joint disease.

Advantageous Effects of Invention

The present invention provides a novel peptide having boneformation-promoting effect and chondrocyte growth-promoting effect. Thepeptide can promote bone formation via oral ingestion, and is thususeful as a medicament, a food or drink product, a supplement, a foodadditive, a cosmetic product, etc. that are for promoting bone formationor for preventing or alleviating a cartilage disorder or joint pain.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a chart showing the measurement results of the osteoblastgrowth-promoting activity of fractions resulting from fractionation ofan egg-yolk protein hydrolysate with a UF membrane with a cut-offmolecular weight of 1 kDa.

FIG. 2 shows a chart showing the measurement results of the osteoblastgrowth-promoting activity of a non-adsorbed fraction (flow-through) andfractions eluted with sodium chloride solutions at concentrations of 750mM and 1 M from an anion-exchange column loaded with a fraction with amolecular weight of 1 kDa or more obtained from the egg-yolk proteinhydrolysate.

FIG. 3 is a chart showing fractions resulting from reverse-phase HPLCfractionation of the 750 mM NaCl eluate fraction, which showed highosteoblast growth-promoting activity.

FIG. 4 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of G1 to G9 indicated in FIG. 3.

FIG. 5 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of fractions 11 to 20 contained in G3 and G4.

FIG. 6 is a chart showing peaks detected in fraction 12 by HPLC underconditions different from those of the above HPLC.

FIG. 7 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of fractions with the two peaks indicated inFIG. 6.

FIG. 8 shows the analysis results of the structure of peptides.

FIG. 9 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of a synthetic peptide (No. 1).

FIG. 10 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of synthetic peptides (No. 2 and No. 3).

FIG. 11 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of synthetic peptides (No. 4 and No. 5).

FIG. 12 is a chart showing the measurement results of the osteoblastgrowth-promoting activity of a synthetic peptide (No. 6).

FIG. 13 is a chart showing the measurement results of the increasedheight of the tibial growth plates of rats after oral administration ofa synthetic peptide (No. 1).

FIG. 14 is a chart showing the measurement results of the chondrocytegrowth-promoting activity of synthetic peptides (No. 1 and No. 6).

FIG. 15 is a chart showing the measurement results of the hyaluronicacid production-promoting activity of synthetic peptides (No. 1 and No.6).

FIG. 16 shows the experimental protocol of Example 14.

FIG. 17 is a chart showing the measurement results of the growth rate ofthe growth cartilage per day (in the longitudinal direction) in asynthetic peptide administration group.

FIG. 18 shows fluorescent microscope images of the formation of primarycancellous bone in a synthetic peptide administration group.

FIG. 19 is a chart showing an increase in the serum level ofinsulin-like growth factor (IGF-1) in a synthetic peptide administrationgroup.

FIG. 20 shows the experimental protocol of Example 15.

FIG. 21 is a chart showing the calculation results of the increasedheight of the rat tibiae per day in a synthetic peptide administrationgroup.

FIG. 22 is a chart showing the measurement results of the secondarycancellous bone volume relative to the bone tissue volume in rats in asynthetic peptide administration group.

FIG. 23 is a chart showing the measurement results of the percentage ofthe bone surface occupied by osteoblasts relative to the total bonesurface in a synthetic peptide administration group.

FIG. 24 is a chart showing the measurement results of the percentage ofthe bone surface occupied by osteoclasts relative to the total bonesurface in a synthetic peptide administration group.

FIG. 25 shows the experimental protocol of Example 16.

FIG. 26 is a chart showing the calculation results of the mineralapposition rate, per day, in the secondary cancellous bone of rats in asynthetic peptide administration group.

FIG. 27 is a chart showing the measurement results of the percentage ofthe bone surface occupied by osteoblasts relative to the total bonesurface in a synthetic peptide administration group.

FIG. 28 is a chart showing the measurement results of the number ofosteoblasts relative to the bone tissue volume in rats in a syntheticpeptide administration group.

FIG. 29 is a chart showing the calculation results of the bone formationrate in a synthetic peptide administration group.

Description of Embodiments

Peptides

The present invention provides a peptide having osteoblastgrowth-promoting activity.

The inventors investigated the identification of peptides with strongactivity for promoting the growth of osteoblasts from a peptide mixtureobtained by hydrolysis of egg yolk proteins, and isolated the peptidesconsisting of the amino acid sequences (i) to (vi) shown below. Theamino acid sequences (ii) to (vi) are partial sequences of the aminoacid sequence (i).

(i) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu (ii) (SEQ ID NO: 2) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu (iii)(SEQ ID NO: 3) Val-Asn-Pro-Glu-Ser-Glu-Glu (iv) (SEQ ID NO: 4)Pro-Glu-Ser-Glu-Glu (v) (SEQ ID NO: 5) Asp-Glu-Ser-Ser-Pro-Tyr-Glu (vi)(SEQ ID NO: 6) Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu

The peptide provided by the present invention is a peptide havingosteoblast growth-promoting activity, comprising an amino acid sequenceselected from

(a) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu,

(b) an amino acid sequence derived from the amino acid sequence (a) byconservative substitution or deletion of 1 to 3 amino acids, and

(c) an amino acid sequence consisting of at least four contiguous aminoacids of the amino acid sequence (a) or (b).

The peptide of the present invention may be a peptide consisting of theamino acid sequence (a), (b) or (c), or a peptide comprising the aminoacid sequence (a), (b) or (c) and an amino acid sequence other than (a),(b) or (c). In cases where the peptide of the present inventioncomprises an amino acid sequence other than (a), (b) or (c), the aminoacid sequence other than (a), (b) or (c) is not particularly limited aslong as the peptide retains osteoblast growth-promoting activity. Theamino acid sequence other than (a), (b) or (c) may be, for example, atag sequence (e.g., a polyhistidine tag, a Myc tag, a FLAG tag, etc.).

The number of the amino acid residues contained in the peptide of thepresent invention is not particularly limited, but is preferably 1000 orless, more preferably 500 or less, further more preferably 200 or less,further more preferably 100 or less, further more preferably 80 or less,further more preferably 70 or less, further more preferably 60 or less,further more preferably 50 or less, further more preferably 40 or less,further more preferably or less, further more preferably 20 or less,particularly preferably 15 or less. The minimum number of the amino acidresidues is not particularly limited as long as the peptide retainsosteoblast growth-promoting activity, but the minimum number of theamino acid residues is preferably 3 or more, more preferably 4 or more,further more preferably 5 or more.

A homology search revealed that the amino acid sequence of SEQ ID NO: 1corresponds to positions 1060 to 1074 of the amino acid sequence ofvitellogenin-2 (SEQ ID NO: 7), which is a precursor protein contained inan egg yolk. The peptide of the present invention is, hence, preferablya fragment of vitellogenin-2 that contains at least four contiguousamino acids at positions 1060 to 1074 of vitellogenin-2 and hasosteoblast growth-promoting activity.

Vitellogenin-2 is a precursor protein contained in an egg yolk, and isknown to be cleaved into four yolk proteins: lipovitellin-1, phosvitin,lipovitellin-2 and YGP40 (yolk glycoprotein of 40 kDa) (see the databaseUniProt: P02845 (VIT2 CHICK)). Positions 1060 to 1074 of vitellogenin-2corresponds to positions 1045 to 1059 of the amino acid sequence oflipovitellin-1 (SEQ ID NO: 8). The peptide of the present invention is,hence, preferably a fragment of lipovitellin-2 that contains at leastfour contiguous amino acids at positions 1045 to 1059 of lipovitellin-1and has osteoblast growth-promoting activity.

Preferably, the peptide of the present invention is a peptide consistingof any of the amino acid sequences (i) to (vi).

The term “conservative substitution of amino acids” herein meanssubstitution of an amino acid for another one within the same groupshown in Table 1 below. In Table 1, preferred conservative substitutionsof amino acids include a substitution between aspartic acid and glutamicacid, a substitution among arginine, lysine, and histidine, asubstitution between tryptophan and phenylalanine, a substitutionbetween phenylalanine and valine, a substitution among leucine,isoleucine and alanine, and a substitution between glycine and alanine.

TABLE 1 Acidic amino acids Aspartic acid (D), glutamic acid (E) Basicamino acids Arginine (R), lysine (K), histidine (H) Hydrophilic aminoSerine (S), threonine (T), asparagine (N), acids glutamine (Q)Hydrophobic amino Tryptophan (W), phenylalanine (F), valine (V), acidsleucine (L), isoleucine (I), methionine (M), proline (P), alanine (A)Aromatic amino acids Tyrosine (Y), tryptophan (W), phenylalanine (F)Hydroxyamino acids Serine (S), threonine (T) Sulfur-containing Cysteine(C), cystine, methionine (M) amino acids Small amino acids Glycine (G),alanine (A), serine (S), methionine (M), threonine (T)

The peptide derivative of the present invention is derived from apeptide with a particular amino acid sequence and the C-terminus of thepeptide derivative may be a carboxyl group (—COOH), a carboxylate(—COO⁻), an amide (—CONH₂) or an ester (—COOR). Examples of R of theester include C₁₋₆ alkyl groups such as methyl, ethyl, n-propyl,isopropyl and n-butyl; C₃₋₈ cycloalkyl groups such as cyclopentyl andcyclohexyl; C₆₋₁₂ aryl groups such as phenyl and α-naphthyl; C₇₋₁₄aralkyl groups including phenyl-C₁₋₂ alkyl groups, such as benzyl andphenethyl, and α-naphthyl-C₁₋₂ alkyl groups, such as α-naphthylmethyl;and a pivaloyloxymethyl group, which is commonly used as an ester fororal administration. Examples of the amide include an amide; an amidesubstituted with one or two C₁₋₆ alkyl groups; an amide substituted withone or two C₁₋₆ alkyl groups substituted with a phenyl group; and anamide that forms a 5- to 7-membered azacycloalkane containing thenitrogen atom of the amide group. When the peptide of the presentinvention has a carboxyl group or a carboxylate at a position other thanthe C-terminus, those with amidated or esterified carboxyl orcarboxylate are also included in the peptide derivative of the presentinvention.

The peptide derivative of the present invention also includes thepeptides of the present invention in which the N-terminal amino group isprotected with a protecting group (e.g., a C₁₋₆ acyl group including aformyl group and C₂₋₆ alkanoyl groups such as acetyl), the peptides ofthe present invention in which a N-terminal glutamyl group generated byin vivo cleavage of the N-terminus is converted to a pyroglutamate, andthe peptides of the present invention in which a substituent (e.g., —OH,—SH, an amino group, an imidazole group, an indole group, or a guanidinogroup) on an amino acid side chain in the molecule is protected with asuitable protecting group (e.g., a C₁₋₆ acyl group including a formylgroup and C₂₋₆ alkanoyl groups such as acetyl).

The side chains of the amino acids constituting the peptide derivativeof the present invention may be modified with a substituent. Examples ofthe substituent include, but are not limited to, a fluorine atom, achlorine atom, a cyano group, a hydroxy group, a nitro group, an alkylgroup, a cycloalkyl group, an alkoxy group, an amino group, and aphosphate group. The side-chain substituent may be protected with aprotecting group. The derivative of the peptide of the present inventionalso includes glycopeptides, which are peptides having sugar chains.

The peptide of the present invention or a derivative thereof may form asalt. The salt is preferably physiologically acceptable. Examples of thephysiologically acceptable salt include salts with acids such ashydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaricacid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid,oleic acid, and palmitic acid; salts with hydroxides or carbonates of analkali metal, such as sodium, potassium and calcium, salts withhydroxides or carbonates of an alkaline earth metal, and salts withaluminum hydroxide or carbonate; and salts with triethylamine,benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine,etc.

The peptide of the present invention or a derivative thereof maycomprise a D-amino acid or a non-naturally occurring amino acid to theextent that the peptide or derivative retains its characteristics. Thepeptide of the present invention or a derivative thereof may compriseanother substance linked thereto to the extent that the peptide orderivative retains its characteristics. Examples of the substancelinkable to the peptide include other peptides, lipids, sugars, sugarchains, an acetyl group, and naturally occurring or synthetic polymers.The peptide of the present invention may be subjected to modificationsuch as glycosylation, side-chain oxidation, and phosphorylation to theextent that the resulting modified peptide retains the characteristicsof the original peptide.

The peptide of the present invention or a derivative thereof or a saltthereof preferably has at least one phosphorylated serine. The positionof the phosphorylated serine is not particularly limited, but preferablyat least one of the serine residues at positions 5, 11 and 12 of theamino acid sequence of SEQ ID NO: 1 is phosphorylated, more preferablyat least one of the serine residues at positions 5 and 11 isphosphorylated, and further more preferably the serine residues atpositions 5 and 11 are phosphorylated. Phosphorylation of the serineresidue(s) has been shown to result in an increase in the effect ofpromoting the growth of bone-forming cells.

Preferred peptide derivatives of the present invention are peptides witha phosphorylated serine residue represented by the amino acid sequencesof SEQ ID NOs: 9 to 14 (see FIG. 10).

The peptide of the present invention or a derivative thereof or a saltthereof (hereinafter the peptide, the derivative and the salt arecollectively and simply called “the peptide of the present invention”)can be produced by solid-phase synthesis (the Fmoc or Boc method) orliquid-phase synthesis in accordance with a known standard peptidesynthesis protocol. Alternatively, the peptide of the present inventioncan be produced by using a transformant carrying an expression vectorcontaining a DNA encoding the peptide of the present invention.Alternatively, the peptide of the present invention can be produced bypreparing a peptide using a transformant carrying an expression vectorcontaining a DNA encoding a peptide comprising the peptide of thepresent invention, and cleaving the resulting peptide with a suitableprotease or peptidase. Alternatively, the peptide of the presentinvention can be produced by a method using an in vitrotranscription-translation system.

The peptide of the present invention can be obtained by purifying ahydrolysate of chicken egg yolk proteins. The preparation method of ahydrolysate of egg yolk proteins and the purification method of thepeptide are not particularly limited, and the preparation andpurification may be done by a known method selected as appropriate.Specifically, the peptide can be obtained by, for example, preparing adefatted egg yolk powder, then preparing a hydrolysate from the powderusing an enzyme such as a protease, and purifying a peptide of interestfrom the hydrolysate by ultrafiltration or chromatography such as HPLC.

Whether the peptide of interest has osteoblast growth-promoting activitycan be determined by comparing the growth of osteoblasts between theculture in the presence of the peptide and the culture in the absence ofthe peptide in a test system selected as appropriate from known methodsfor measuring the cell growth. A higher growth level of osteoblasts inthe culture in the presence of the peptide indicates that the peptidehas the promoting activity. Specifically, for example, MTT assay or cellcounting method may be performed in a cell culture system

Whether the peptide of interest has chondrocyte growth-promotingactivity can be determined by comparing the growth of chondrocytes orchondrogenic cells between the culture in the presence of the peptideand the culture in the absence of the peptide in a test system selectedas appropriate from known methods for measuring the cell growth. Ahigher growth level of osteoblasts in the culture in the presence of thepeptide indicates that the peptide has the promoting activity.Specifically, for example, MTT assay or cell counting method may beperformed in a cell culture system using an osteoblast-derived cellline.

Whether the peptide of interest has hyaluronic acid production-promotingactivity can be determined by measuring, by a known method, the amountsof hyaluronic acid production in the culture supernatant in cultureusing cultured cells with an ability to produce hyaluronic acid, andthen comparing the amounts of hyaluronic acid between the culture in thepresence of the peptide and the culture in the absence of the peptide. Ahigher amount of hyaluronic acid in the supernatant of the culture inthe presence of the peptide indicates that the peptide has the promotingactivity. Examples of the measurement method of the amount of hyaluronicacid include ELISA.

Polynucleotides

The polynucleotide provided by the present invention is a polynucleotideencoding the above peptide of the present invention. The polynucleotidecan be present in the form of RNA (e.g., mRNA) or DNA (e.g., cDNA orgenomic DNA). The polynucleotide may be a double or single strand. Thedouble strand may be a double-stranded DNA, a double-stranded RNA or aDNA-RNA hybrid. The single strand may be a coding strand (sense strand)or a non-coding strand (antisense strand). The polynucleotide of thepresent invention may be fused with a polynucleotide encoding a taglabel (a tag sequence or a marker sequence) at the 5′- or 3′-terminus.The polynucleotide of the present invention may contain an untranslatedregion (UTR) sequence, a vector sequence (including an expression vectorsequence), etc.

The nucleotide sequence of the polynucleotide encoding the peptide ofthe present invention can be custom designed by appropriately selectingcodons for amino acids based on the amino acid sequence of the peptideof the present invention and combining the selected codons. As describedabove, the amino acid sequence of SEQ ID NO: 1 is part of the amino acidsequence of vitellogenin-2 (SEQ ID NO: 7), which is a precursor proteincontained in an egg yolk, and therefore the nucleotide sequence of thepolynucleotide can be designed based on the nucleotide sequence of thegene encoding vitellogenin-2.

The polynucleotide of the present invention can be produced by a knownDNA synthesis method, PCR, etc. Specifically, for example, thenucleotide sequence is designed by appropriately selecting codons foramino acids based on the amino acid sequence of the peptide of thepresent invention, and the designed sequence is chemically synthesizedusing a commercially available DNA synthesizer. Alternatively, primersare designed for amplification of the coding region of the peptide ofthe present invention in the nucleotide sequence of the gene encodingvitellogenin-2 (accession No.: X13607), and then PCR etc. are performedwith chicken genomic DNA or cDNA as a template using the designedprimers to produce a DNA fragment containing the polynucleotide of thepresent invention in large quantities.

Expression Vectors

The present invention provides an expression vector used for theproduction of the peptide of the present invention. The expressionvector of the present invention is not particularly limited as long asit contains a polynucleotide encoding the peptide of the presentinvention, but preferred are plasmid vectors carrying a RNA polymeraserecognition sequence (pSP64, pBluescript, etc.). The method forpreparing the expression vector is not particularly limited, and theexpression vector may be prepared with the use of a plasmid, a phage, acosmid or the like. The type of the vector is not particularly limitedand any appropriate vector that can be expressed in host cells can beselected. For example, a promoter sequence is selected as appropriatefor the type of host cells to ensure the expression of thepolynucleotide of the present invention, and this promoter sequence andthe polynucleotide of the present invention are inserted into a plasmidetc. to give a desired expression vector. After a host transformed withthe expression vector of the present invention is cultured, cultivatedor bred, the peptide of the present invention can be collected andpurified from the culture products etc. by conventional methods (e.g.,filtration, centrifugation, cell disruption, gel filtrationchromatography, ion exchange chromatography, etc.).

The expression vector preferably contains at least one selection marker.Examples of the marker include a dihydrofolate reductase gene and aneomycin resistance gene for eukaryote cell culture; and a tetracyclineresistance gene and an ampicillin resistance gene for culture ofEscherichia coli and other bacteria. Such a selection marker is usefulfor checking whether the polynucleotide of the present invention hasbeen successfully transfected into host cells and whether thepolynucleotide is reliably expressed therein. Alternatively, the peptideof the present invention may be expressed as a fusion protein. Thepeptide of the present invention may be expressed as, for example, a GFPfusion protein using green fluorescent protein (GFP) of Aequoreavictoria as a marker.

The host is not particularly limited and various known cells cansuitably be used. Specific examples of the cells include bacteria suchas Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae andfission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditiselegans), Xenopus laevis oocytes and animal cells (e.g., CHO cells, COScells and Bowes melanoma cells). The method for transfecting host cellswith the expression vector, i.e. the transformation method, is also notparticularly limited and known methods can suitably be used, includingelectroporation, the calcium phosphate method, the liposome method andthe DEAE dextran method.

Transformants

The present invention provides a transformant carrying the expressionvector of the present invention. As used herein, the transformantencompasses a cell, a tissue and an organ as well as an individualorganism. The type of the organism to be transformed is not particularlylimited, and examples thereof include various microorganisms, plants andanimals listed above as examples of the host cells. The transformant ofthe present invention expresses the peptide of the present invention. Itis preferred that the transformant of the present invention stablyexpresses the peptide of the present invention, but the transformant maytransiently express the peptide.

Bone Formation Promoter, Bone Resorption Inhibitor, Chondrocyte GrowthPromoter, Hyaluronic Acid Production Promoter, Chondrogenic CellDifferentiation Inducer, Mesenchymal Stem Cell Growth Promoter, andMesenchymal Stem Cell Differentiation Inducer

The present invention provides a bone formation promoter comprising thepeptide of the present invention. Since the peptide of the presentinvention has bone formation-promoting activity, the peptide is suitableas an active ingredient for a bone formation promoter. The boneformation-promoting activity of the peptide of the present invention canbe confirmed by, for example, administering (e.g., oral administering)the peptide of the present invention to an animal subject for a certainperiod of time (e.g., for 7, 14 or 21 days), and measuring an indicatorfor the activity, including an enhanced increase in the height of thetibial growth plate, an increase in the growth rate of the growthcartilage, promotion of the formation of the primary cancellous bone, anincrease in the serum level of insulin-like growth factor (IGF-1), anincrease in the secondary cancellous bone volume relative to the bonetissue volume, an increase of osteoblasts, a reduction of osteoclasts,an increase in the mineral apposition rate in the secondary cancellousbone, etc. The term “growth cartilage” herein refers to, for example,the growing area of the bone at the epiphysis. The term “primarycancellous bone” refers to, for example, immature cancellous boneimmediately below the growth cartilage. The term “secondary cancellousbone” refers to, for example, mature cancellous bone. The term“promotion of bone formation” herein can be understood as “an enhancedincrease in the height of the tibial growth plate”, “an increase in thegrowth rate of the growth cartilage”, “promotion of the formation of theprimary cancellous bone”, “an increase in the serum level ofinsulin-like growth factor (IGF-1)”, “an increase in the secondarycancellous bone volume relative to the bone tissue volume”, “an increaseof osteoblasts”, “a reduction of osteoclasts”, “an increase in themineral apposition rate in the secondary cancellous bone”, etc. Thepresent invention also provides an osteoblast growth promotor comprisingthe peptide of the present invention. Since the peptide of the presentinvention has osteoblast growth-promoting activity, the peptide issuitable as an active ingredient for an osteoblast growth promotor. Thepresent invention also provides a bone resorption inhibitor comprisingthe peptide of the present invention. The peptide of the presentinvention has bone resorption-inhibiting activity, and is thus suitableas an active ingredient for a bone resorption inhibitor. The presentinvention also provides a chondrocyte growth promotor comprising thepeptide of the present invention. Since the peptide of the presentinvention has chondrocyte growth-promoting activity, the peptide issuitable as an active ingredient for a chondrocyte growth promoter. Thepresent invention also provides a hyaluronic acid production promotercomprising the peptide of the present invention. Since the peptide ofthe present invention has hyaluronic acid production-promoting activity,the peptide is suitable as an active ingredient for a hyaluronic acidproduction promoter. The present invention also provides a chondrogeniccell differentiation inducer comprising the peptide of the presentinvention. Since the peptide of the present invention hasdifferentiation inducing activity on chondrogenic cells, the peptide issuitable as an active ingredient for a chondrogenic cell differentiationinducer. The present invention also provides a mesenchymal stem cellgrowth promoter comprising the peptide of the present invention. Thepeptide of the present invention has growth-promoting activity onmesenchymal stem cells, and is thus suitable as an active ingredient fora mesenchymal stem cell growth promoter. The present invention alsoprovides a mesenchymal stem cell differentiation inducer comprising thepeptide of the present invention. The peptide of the present inventionhas differentiation inducing activity on mesenchymal stem cells, and isthus suitable as an active ingredient for a mesenchymal stem celldifferentiation inducer.

In addition to the peptide of the present invention, lipovitellin-1 or aderivative thereof or a salt thereof is also suitable as an activeingredient for the above promoter, inhibitor or inducer. Thelipovitellin-1 herein refers to a protein that consists of an amino acidsequence identical or substantially identical to the amino acid sequenceof SEQ ID NO: 8 and has substantially the same activity as that oflipovitellin-1. The amino acid sequence substantially identical to theamino acid sequence of SEQ ID NO: 8 is, for example, an amino acidsequence derived from SEQ ID NO: 8 by deletion, substitution or additionof one to several amino acids. The phrase “deletion, substitution oraddition of one to several amino acids” means deletion, substitution oraddition of amino acids whose number is substantially equal to thenumber of the residues that can be deleted, substituted or added by aknown method for mutagenesis of peptides, such as site-directedmutagenesis (preferably 10 amino acids or less, more preferably 7 aminoacids or less, and even more preferably 5 amino acids or less). Such amutant protein is not limited to a protein artificially mutated by aknown method for mutagenesis of polypeptides, and may be a proteinisolated and purified from nature. In addition, the amino acid sequencesubstantially identical to the amino acid sequence of SEQ ID NO: 1 is,for example, an amino acid sequence that is at least 80% identical, morepreferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 1.

A derivative of lipovitellin-1 is preferably those listed above as theexamples of the derivative of the peptide of the present invention. Asalt of lipovitellin-1 or of a derivative of lipovitellin-1 ispreferably those listed above as the examples of the salt of the peptideof the present invention or of the derivative of the peptide of thepresent invention.

Lipovitellin-1, a derivative thereof and a salt thereof can be producedas a recombinant protein by known genetic modification techniques or bymethods using in vitro transcription-translation system. Alternatively,lipovitellin-1, a derivative thereof and a salt thereof can be purifiedfrom chicken egg yolk proteins.

Hereinafter, the term “peptide etc. of the present invention”encompasses lipovitellin-1, a derivative thereof and a salt thereof inaddition to “the peptide of the present invention”.

The peptide etc. of the present invention have been shown to exhibit thedesired effects through oral administration to a mammal, and thus theabove promoters, inhibitors and inducers are suitable for oraladministration. The peptide etc. of the present invention, which are theegg yolk protein lipovitellin-1 or a fragment thereof, are highly safeand have mild effects, and thus can be administered or used for a longperiod of time.

Medicaments

The present invention provides a medicament comprising the peptide etc.of the present invention. Since the peptide etc. of the presentinvention have osteoblast growth-promoting activity, the peptide etc.can be used as a medicament for promoting bone formation. The medicamentfor promoting bone formation of the present invention can be suitable asa medicament for preventing or treating, for example, osteoporosis,osteogenesis imperfecta, hypercalcemia, hyperparathyroidism,osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis,Paget's disease, rheumatoid arthritis, bone loss due to osteoarthritis,inflammatory arthritis, osteomyelitis, glucocorticoid treatment,metastatic bone disease, periodontal bone loss, cancerous bone loss,age-related bone loss, fracture, and low back pain.

Since the peptide etc. of the present invention have chondrocytegrowth-promoting activity and/or hyaluronic acid production-promotingactivity, the peptide etc. can be used as a medicament for preventing oralleviating a cartilage disorder or a joint disease. Examples of thecartilage disorder include osteoarthritis, cartilage defects, cartilageinjury, and meniscus injury. Examples of the joint disease include jointpain, rheumatoid arthritis, osteoarthritis, suppurative arthritis, goutyarthritis, traumatic arthritis, and degenerative joint disease.

The medicament of the present invention can be produced by appropriatelyblending the peptide etc. of the present invention as an activeingredient with a pharmaceutically acceptable carrier or additive inaccordance with a known production method for pharmaceuticalpreparations (e.g., the methods described in the Japanese pharmacopoeia,etc.). In particular, the medicament may be, for example, an oralpreparation or a parenteral preparation, including tablets (includingsugar-coated tablets, film-coated tablets, sublingual tablets, orallydisintegrating tablets, and buccal tablets), pills, powders, granules,capsules (including soft capsules and microcapsules), troches, syrups,liquids, emulsions, suspensions, controlled-release preparations (e.g.,fast-release preparations, sustained release preparations, and sustainedrelease microcapsules), aerosols, films (e.g., orally disintegratingfilms, and oral mucosal adhesive films), injections (e.g., subcutaneousinjections, intravenous injections, intramuscular injections, andintraperitoneal injections), intravenous infusions, transdermalpreparations, ointments, lotions, patches, suppositories (e.g., rectalsuppositories and vaginal suppositories), pellets, transnasalpreparations, transpulmonary preparations (inhalants), and eye drops.The amount of the carrier or additive to be added can be determined asappropriate based on the range typically used in the pharmaceuticalfield. The carrier or additive that may be added is not particularlylimited and examples thereof include various types of carriers such aswater, physiological saline, other aqueous solvents, and aqueous or oilyvehicles; and various types of additives such as excipients, binders, pHadjusters, disintegrants, absorption promoters, lubricants, colorants,flavors and fragrances.

Examples of the additives that may be added to tablets, capsules, etc.include binders such as gelatin, corn starch, tragacanth, and gumarabic; excipients such as crystalline cellulose; swelling agents suchas corn starch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, and saccharin; andflavors such as peppermint flavor, wintergreen oil, and cherry flavor.When the unit dosage form is a capsule, a liquid carrier such as oilsand fats can be further added in addition to the above types ofmaterials. A sterile composition for injection can be prepared inaccordance with a usual pharmaceutical practice (for example, bydissolving or suspending the active ingredient in a solvent such aswater for injection or a natural vegetable oil). Aqueous liquids forinjection that may be used are, for example, physiological saline and anisotonic solution containing glucose and/or other auxiliary substances(for example, D-sorbitol, D-mannitol, sodium chloride, etc.). Theaqueous liquids for injection may be used in combination with anappropriate solubilizer, such as alcohols (e.g., ethanol), polyalcohols(e.g., propylene glycol, polyethylene glycol, etc.), and nonionicsurfactants (e.g., polysorbate 80™, HCO-50, etc.). Oily liquids that maybe used are, for example, sesame oil and soybean oil. The oily liquidsmay be used in combination with a solubilizer such as benzyl benzoateand benzyl alcohol. Other additives that may be added are, for example,buffering agents (e.g., a phosphate buffer, a sodium acetate buffer,etc.), soothing agents (e.g., benzalkonium chloride, procainehydrochloride, etc.), stabilizers (e.g., human serum albumin,polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol,etc.) and antioxidants.

The preparations produced in the above manner are safe and have lowtoxicity, and thus can be administered to, for example, humans and othermammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs,monkeys, etc.).

The amount of the peptide etc. of the present invention in themedicament of the present invention may vary with the dosage form, theadministration method, the carrier to be used, etc., but is usually 0.01to 100% (w/w), preferably 0.1 to 95% (w/w), relative to the total amountof the preparation. The medicament of the present invention containingthe peptide etc. of the present invention in such an amount can beproduced in accordance with a conventional method.

The dosage varies with the subject to which the medicament is to beadministered, the symptom, the route of administration, etc., but ingeneral, the dosage for oral administration to a human with a bodyweight of about 60 kg is about 0.01 to 1000 mg per day, preferably about0.1 to 100 mg per day, and more preferably about 0.5 to 500 mg per day.The total daily dosage may be administered in a single dose or individed doses.

The medicament of the present invention is expected to exhibit a highadditive or synergistic effect in combination with another medicamentfor treating a bone disease or for treating a cartilage or jointdisease. Said another medicament for promoting bone formation may be,for example, an active vitamin D₃ preparation, a vitamin K₂ preparation,a parathyroid hormone preparation (teriparatide), a female hormonepreparation (estrogen), a bisphosphonate preparation, a SERM (raloxifenehydrochloride), a calcitonin preparation, etc. Said another medicamentfor treating a cartilage or joint disease may be, for example,glucosamine, chondroitin, type I collagen, type II collagen,N-acetylglucosamine, etc.

Food or Drink Products

The present invention provides a food or drink product comprising thepeptide etc. of the present invention. The food or drink product of thepresent invention is suitable as a food or drink product for promotingbone formation and as a food or drink product for preventing and/oralleviating a cartilage disorder or a joint disease.

The food or drink product includes health foods, functional foods, foodsfor specified health use, and foods for sick people. The form of thefood or drink product is not particularly limited and examples thereofinclude drinks such as tea drink, refreshing drink, carbonated drink,nutritional drink, fruit juice, and lactic drink; noodles such asbuckwheat noodle, wheat noodle, Chinese noodle, and instant noodle;sweets and bakery products such as drop, candy, gum, chocolate, snack,biscuit, jelly, jam, cream, pastry, and bread; fishery and livestockproducts such as fish sausage, ham, and sausage; dairy products such asprocessed milk and fermented milk; fats, oils, and processed foodsthereof, such as vegetable oil, oil for deep frying, margarine,mayonnaise, shortening, whipped cream, and dressing; seasonings such assauce and dipping sauce; retort pouch foods such as curry, stew,rice-bowl cuisine, porridge, and rice soup; and frozen desserts such asice cream, sherbet, and shaved ice.

The present invention also provides a supplement comprising the peptideetc. of the present invention. The supplement of the present inventionis suitable as a supplement for promoting bone formation and as asupplement for preventing and/or alleviating a cartilage disorder or ajoint disease. The supplement can be provided in the form of, forexample, tablets, granules, powders or drinks.

The present invention provides a food additive comprising the peptideetc. of the present invention. The food additive of the presentinvention is suitable as a food additive for promoting bone formationand as a food additive for preventing and/or alleviating a cartilagedisorder or a joint disease. The form of the food additive of thepresent invention is not particularly limited, and may be, for example,a liquid, a paste, a powder, flakes, granules, etc. The food additive ofthe present invention can be produced in accordance with a conventionalproduction method for food additives.

Also provided is a feed or a feed additive comprising the peptide etc.of the present invention.

The present invention provides a cosmetic product comprising the peptideetc. of the present invention. The cosmetic product of the presentinvention is suitable as a cosmetic product for promoting bone formationand as a cosmetic product for preventing and/or alleviating a cartilagedisorder or a joint disease. The cosmetic product includes the so-calledmedicated cosmetics (quasi drugs). Examples of the cosmetic productinclude washing lotions, shampoos, rinses, hair tonics, hair lotions,aftershave lotions, body lotions, makeup lotions, cleansing creams,massage creams, emollient creams, aerosol products, deodorizers,fragrances, deodorants, and bath fragrances. Depending on the purpose,the cosmetic product of the present invention may contain a componentgenerally used in cosmetic products in addition to the peptide of thepresent invention, and such a component includes, for example,surfactants, moisturizers, animal- and plant-derived fats and oils,silicones, higher alcohols, lower alcohols, animal- and plant-derivedextracts, ultraviolet absorbers, anti-inflammatories, sequesteringagents, vitamins, antioxidants, thickeners, preservatives, bactericides,pH adjusters, colorants, and various fragrances.

The present invention further includes the following.

(a) Use of the peptide of the present invention or a derivative thereofor a salt thereof or lipovitellin-1 or a derivative thereof or a saltthereof for the production of a medicament for promoting bone formationor for preventing or alleviating a cartilage disorder or a jointdisease.

(b) The peptide of the present invention or a derivative thereof or asalt thereof or lipovitellin-1 or a derivative thereof or a salt thereoffor use in the promotion of bone formation or for the prevention oralleviation of a cartilage disorder or a joint disease.

(c) A method for promoting bone formation, the method comprisingadministering, to a mammal, an effective amount of the peptide of thepresent invention or a derivative thereof or a salt thereof orlipovitellin-1 or a derivative thereof or a salt thereof.

(d) A method for preventing or alleviating a cartilage disorder or ajoint disease, the method comprising administering, to a mammal, aneffective amount of the peptide of the present invention or a derivativethereof or a salt thereof or lipovitellin-1 or a derivative thereof or asalt thereof.

(e) A non-therapeutic method for promoting bone formation, the methodcomprising orally administering, to a mammal, the peptide of the presentinvention or a derivative thereof or a salt thereof or lipovitellin-1 ora derivative thereof or a salt thereof.

(f) A non-therapeutic method for preventing or alleviating a cartilagedisorder or a joint disease, the method comprising orally administering,to a mammal, the peptide of the present invention or a derivativethereof or a salt thereof or lipovitellin-1 or a derivative thereof or asalt thereof.

(g) The peptide of the present invention or a derivative thereof or asalt thereof or lipovitellin-1 or a derivative thereof or a salt thereoffor use in the promotion of bone formation or for the prevention oralleviation of a cartilage disorder or a joint disease.

(h) Use of the peptide of the present invention or a derivative thereofor a salt thereof or lipovitellin-1 or a derivative thereof or a saltthereof for the promotion of bone formation or for the prevention oralleviation of a cartilage disorder or a joint disease.

(i) Use of the peptide of the present invention or a derivative thereofor a salt thereof or lipovitellin-1 or a derivative thereof or a saltthereof for the production of a medicament for promoting bone formationor for preventing or alleviating a cartilage disorder or a jointdisease.

EXAMPLES

The present invention will be described in more detail below withreference to Examples, but the present invention is not limited thereto.

Example 1 Identification of Active Substances for Promoting Growth ofOsteoblasts in Egg-Yolk Protein Hydrolysate

(1) Preparation of Egg-Yolk Protein Hydrolysate

To 100 parts by mass of defatted egg yolk powder (Kewpie Corporation),500 parts by mass of water was added, and the mixture was stirred untiluniform. To the mixture, 5 parts by mass of “Alcalase” (trade name)(protease from Bacillus licheniformis (Novozymes Japan Ltd.)) was added,and the mixture was allowed to react at pH 7 at 55 to 60° C. for 3hours. At the end of the reaction, the enzyme was inactivated by heatingat 90° C. for 10 minutes. Filtration was performed and the filtrate wascollected. The filtrate was spray-dried to give an egg-yolk proteinhydrolysate.

Instead of the defatted egg yolk powder as used above, a defatted eggyolk powder prepared from a commercially available egg yolk powder(e.g., a product produced by Kewpie Corporation) may be used.Specifically, 5 to 10 parts by mass of ethanol (or n-hexane) is added to1 part by mass of a commercially available egg yolk powder, then themixture is stirred with a blender for about 30 minutes and filtered, andthe solid is collected. This procedure is repeated about 3 times forremoval of the fat from the egg yolk. The defatted egg yolk is thenair-dried to give a defatted dry powder.

(2) Measurement Method of Osteoblast Growth-Promoting Activity

A mouse osteoblast-like cell line MC3T3-E1 Subclone-4 (ATCC No.CRL-2593) was cultured in α-MEM medium supplemented with 10% FBS at 5%CO₂ and 95% air at 37° C. until confluent. The cells were collected bytrypsinization. The collected cells were suspended at 1×10⁴ cells/mL infresh α-MEM medium. One mL of the cell suspension was seeded in eachwell of a 24-well plate, and precultured at 5% CO₂ and 95% air at 37° C.On the next day, the medium was replaced with medium prepared by wellmixing 50 μL of a sample or PBS (−) with 950 μL of fresh α-MEM mediumsupplemented with CaCl₂ to a Ca concentration of 500 μg/mL. The cellswere further cultured for 72 hours. The growth activity of osteoblastswas measured by the cell counting method. The activity of the sample forpromoting the growth of osteoblasts was expressed in terms of therelative value, with the growth rate in the PBS (−) group taken as 100.

(3) Fractionation with UF Membrane

In 600 mL of water, 60 g of the egg-yolk protein hydrolysate wasdissolved. The solution was fractionated with a UF membrane (cut offmolecular weight: 1 kDa, Nihon Millipore, Ltd.). Osteoblastgrowth-promoting activity was determined for a fraction with a molecularweight of 1 kDa or less and a fraction with a molecular weight of 1 kDaor more. As a positive control, the unfractionated egg-yolk proteinhydrolysate (1 mg/mL) was used. The fraction with a molecular weight of1 kDa or less and the fraction with a molecular weight of 1 kDa or morewere used in amounts equal to the corresponding amounts in theunfractionated egg-yolk protein hydrolysate (i.e., the correspondingamounts in 0.2 mg of the egg-yolk protein hydrolysate).

The results are shown in FIG. 1. As is apparent from FIG. 1, thefraction with a molecular weight of 1 kDa or more showed osteoblastgrowth-promoting activity.

(4) Fractionation with Anion-Exchange Column

The fraction with a molecular weight of 1 kDa or more, which showedosteoblast growth-promoting activity in the above (3), was furtherfractionated on an anion-exchange column (resin: Dowex, column size: 10mm in diameter×63 mm) to give a flow-through fraction and fractionseluted with aqueous solutions with NaCl concentrations of 750 mM and 1M. Osteoblast growth-promoting activity was determined for thefractions. As a positive control, the unfractionated egg-yolk proteinhydrolysate (1 mg/mL) was used. The fractions were used in amounts equalto the corresponding amounts in the unfractionated egg-yolk proteinhydrolysate (i.e., the corresponding amounts in 10 mg of the egg-yolkprotein hydrolysate).

The results are shown in FIG. 2. As is apparent from FIG. 2, the 750 mMNaCl eluate fraction showed the highest osteoblast growth-promotingactivity.

(5) Fractionation by HPLC (First Round)

The 750 mM NaCl eluate fraction, which showed the highest osteoblastgrowth-promoting activity in the above (4), was further fractionated ona C18 reverse-phase column (column size: 50 mm in diameter×250 mm). Thefractions were collected at 1 minute intervals from elution time zero to45 minute. The HPLC conditions were as follows.

-   -   Column: C18 reverse-phase column,        -   size: 50 mm in diameter×250 mm (HiPep Laboratories)    -   Mobile phase: (A) 10% acetonitrile/0.1% TFA        -   (B) 60% acetonitrile/0.1% TFA        -   B 5%→95% (40 min) gradient    -   Detection: UV 215 nm    -   Amount of samples: 601 mg

In total, 45 fractions were collected, and every five fractions werecombined to give G1 to G9 fractions (see FIG. 3). Osteoblastgrowth-promoting activity was determined for G1 to G9 fractions. Thefractions were used in amounts equal to the corresponding amounts in theunfractionated egg-yolk protein hydrolysate (i.e., the correspondingamounts in 66 mg of the egg-yolk protein hydrolysate). As a positivecontrol, the unfractionated egg-yolk protein hydrolysate (1 mg/mL) wasused.

The results are shown in FIG. 4. As is apparent from FIG. 4, G3 showedthe highest osteoblast growth-promoting activity.

Osteoblast growth-promoting activity was further determined for the fivefractions (fractions 11 to 15) of G3 and the five fractions (fractions16 to 20) of G4. The fractions were used in amounts equal to thecorresponding amounts in the unfractionated egg-yolk protein hydrolysate(i.e., the corresponding amounts in 66 mg of the egg-yolk proteinhydrolysate). As a positive control, the unfractionated egg-yolk proteinhydrolysate (1 mg/mL) was used.

The results are shown in FIG. 5. As is apparent from FIG. 5, allfractions 11 to 20 showed higher osteoblast growth-promoting activitythan the control.

(6) Fractionation by HPLC (Second Round)

HPLC was performed on fractions 11 to 20 under the following conditions.

-   -   Column: ODS-A (trade name) (10×4.6 mm I.D.) (YMC Co., Ltd.)    -   Mobile phase: 4% acetonitrile/0.1% TFA (isocratic)    -   Temperature: 40° C.    -   Detection: UV 215 nm    -   Amount of samples: 50 μg

Of fractions 11 to 20, two distinct peaks were detected in fraction 12(see FIG. 6), and accordingly fraction 12 was further investigated.Briefly, fraction 12 was subjected to HPLC under the above conditions,and fractions with peak 1 and peak 2 were separated (see FIG. 6).Osteoblast growth-promoting activity was determined for the fractionwith peak 1 (peak 1), the fraction with peak 2 (peak 2), and a fractionother than peak 1 and peak 2 fractions (Others). The fractions were usedin amounts equal to the corresponding amounts in the unfractionatedegg-yolk protein hydrolysate (i.e., the corresponding amounts in 133 mgof the egg-yolk protein hydrolysate). As a positive control, theunfractionated egg-yolk protein hydrolysate (1 mg/mL) was used.

The results are shown in FIG. 7. As is apparent from FIG. 7, thefraction with peak 2 showed the highest osteoblast growth-promotingactivity.

(7) Identification of Peptides

The amino acid sequences of the peptides contained in the fraction withpeak 2 were analyzed using MALDI-TOF-MS (mass spectrometer). Theanalysis results revealed that at least six types of peptides werecontained in the fraction with peak 2 as shown in FIG. 8. These peptidesare fragments of lipovitellin-1 contained in egg yolk, and eachcorrespond to the following positions in the amino acid sequence oflipovitellin-1 (SEQ ID NO: 8): No. 1, positions 1045-1052; No. 2,positions 1045-1051; No. 3, positions 1047-1051; No. 4, positions1053-1059; No. 5, positions 1052-1059; and No. 6, positions 1045-1059.

Example 2 Study of Osteoblast Growth-Promoting Activity of SyntheticPeptides

The six types of peptides shown in Example 1 to have osteoblastgrowth-promoting activity were chemically synthesized and theirosteoblast growth-promoting activities were determined. The peptidesynthesis was performed with a SyroII automatic peptide synthesizer(Biotage Japan Ltd.).

Osteoblast growth-promoting activity was determined for the syntheticpeptides in the same manner as in Example 1. The synthetic peptides wereadded at 2.5, 5 or 10 μg/mL per well. As a control, PBS (−) was added.The osteoblast growth-promoting activity was expressed in terms of therelative value, with the growth rate in the PBS (−) group taken as 100.

FIG. 9 shows the results of No. 1, FIG. 10 shows the results of No. 2and No. 3, FIG. 11 shows the results of No. 4 and No. 5, and FIG. 12shows the results of No. 6. As is apparent from FIGS. 9 to 12, all thesynthetic peptides promoted the growth of osteoblasts in aconcentration-dependent manner.

Example 3 Study of Bone Growth Promoting Effect of Synthetic Peptides inAdolescent Rats

One of the synthetic peptides (No. 1) or physiological saline (control)was administered to Sprague-Dawley male rats at three weeks old by oralgavage using a gastric tube once a day for four days. The dose of thesynthetic peptide was 1, 10 or 100 mg/kg·day. As a positive control,human growth hormone (hGH) (Norditropin, Standard CommodityClassification No. of Japan: 872412) was subcutaneously administered at20 μg/k·day once a day for four days. After the administration on day 4,calcein was subcutaneously administered, and the rats were kept for oneday. The rats were euthanized by bloodletting under anesthesia, and thetibiae were harvested. The tissue specimens of the tibial growth plateswere prepared, and calcein deposition areas (fluorescent regions) on thetibial growth plates were measured under a fluorescent microscope todetermine the longitudinal bone growth.

The results are shown in FIG. 13. As is apparent from FIG. 13, theincrease in the height of the tibial growth plates was enhanced in amanner consistent with the increase in the dose of the syntheticpeptide. The effect of enhancing the increase in the height by thesynthetic peptide at the highest dose was comparable to the effect ofthe subcutaneous administration of the human growth hormone. The resultsrevealed that oral administration of the peptides of the presentinvention promotes bone formation.

Example 4 Study of Chondrocyte Growth-Promoting Activity of SyntheticPeptides

This study was performed using mouse-derived chondrogenic cell lineATDC5 (RIKEN BANK, RBC0565). ATDC5 cells in the logarithmic growth phasewere suspended at 3×10⁴ cells/mL in Eagle MEM medium supplemented with5% FCS (fetal calf serum), and 100 μL of the cell suspension was seededin each well of a 96-well plate and precultured at 5% CO₂ and 95% air at37° C. On the next day, the medium in each well was removed, and mediumcontaining the synthetic peptide (No. 1 or No. 6) at 10 nmol/mL wasadded. The cells were cultured for 72 hours. As a control, mediumwithout a synthetic peptide was added. The reaction solution (10μg/well) of Cell Counting Kit-8 (Dojindo Laboratories) was added to eachwell. The plate was incubated for 3 hours, and the absorbance at 450 nmwas measured with a microplate reader. The activity of the syntheticpeptides for promoting the growth of chondrocytes was expressed in termsof the relative value, with the absorbance (growth rate) in the controlgroup taken as 100%.

The results are shown in FIG. 14. As is apparent from FIG. 14, syntheticpeptide No. 6 and synthetic peptide No. 1 (a fragment of No. 6) showedchondrocyte growth-promoting effect.

Example 5 Study of Hyaluronic Acid Production-Promoting Activity ofSynthetic Peptides

This study was performed using the mouse-derived cell line ATDC5 as usedin Example 4. ATDC5 cells in the logarithmic growth phase were seeded at2×10⁴ cells/mL per well in a 24-well plate containing DMEM/F-12 (1:1)medium supplemented with 5% FCS (fetal calf serum), 10 μg/mL transferrinand 3×10⁻⁸ M sodium selenite. The cells were precultured at 5% CO₂ and95% air at 37° C. On the next day, the medium in each well was removed,and medium containing the synthetic peptide (No. 1 or No. 6) at 10nmol/mL was added. The cells were cultured for 72 hours. As a control,medium without a synthetic peptide was added. After 48 hours of culture,the culture supernatants were collected, and their hyaluronic acidconcentrations were measured using QnE Hyaluronic Acid ELISA Assay(Biotech Trading Partners). The concentrations of hyaluronic acid in theculture supernatants of the synthetic peptide addition group werecalculated by subtracting the absorbance of the background from theabsorbance of the supernatants, and fitting the resulting values to thereference curve prepared using the standard.

The results are shown in FIG. 15. As is apparent from FIG. 15, syntheticpeptide No. 6 and synthetic peptide No. 1 (a fragment of No. 6) showedhyaluronic acid production-promoting effect.

Example 6 Bone Density Recovery Test Using Synthetic Peptides

Sprague-Dawley male rats at three weeks old were initially fed with alow-calcium diet (containing 0.05% calcium carbonate) for one week, andthen fed with a normal diet (containing 1.2% calcium carbonate). Thesynthetic peptide (No. 1) or physiological saline (control) wasadministered to the rats by oral gavage using a gastric tube once a dayfor seven days. The dose of the synthetic peptide was 1, 10 or 100mg/kg·day. After the oral administration on day 7, the rats wereeuthanized by bloodletting under anesthesia, and the femurs and tibiaewere harvested. The bone volume and bone morphology of the tibiae andfemurs were measured by dual energy x-ray absorptiometry (DXA) andperipheral quantitative computed tomography (pQCT). The results revealedthat the length, weight, and bone density of the tibiae tended to behigher in the synthetic peptide administration group than in the controlgroup, indicating that the synthetic peptide promoted the recovery ofthe bone density loss due to calcium deficiency.

Example 7 Study Using Postmenopausal Osteoporosis Model ofOvariectomized Rats

Female rats (eight months old, ten rats in each group) underwentovariectomy (OVX), and synthetic peptide No. 1 was administered to therats by oral gavage using a gastric tube once a day from the firstpostoperative day for six months. The dose of the synthetic peptide was1, 10 or 100 mg/kg·day. The results revealed that the OVX-syntheticpeptide administration group showed an increase in the bone density ofthe distal femoral metaphysis in a manner consistent with the increasein the dose of the synthetic peptide, and the increase was significantas compared with the OVX-control group (non-synthetic peptideadministration group). The medium-dose administration group (10mg/kg·day) and the high-dose administration group (100 mg/kg·day) showedan equal or higher bone density as compared with the sham operationgroup without removal of the ovary. Regarding the measured bonemorphology parameters, the cancellous bone volume in the proximal tibialmetaphysis increased in the OVX-synthetic peptide administration groupin a manner consistent with the increase in the dose, and the increasewas significant as compared with the OVX-control group. The medium-doseadministration group and the high-dose administration group showed anequal or higher volume of the cancellous bone as compared with the shamoperation group without removal of the ovary. OVX-induced reduction inthe trabecular numbers and the trabecular width and OVX-induced increasein the trabecular separation tended to be inhibited in the OVX-syntheticpeptide administration group.

Example 8 Bone Formation-Promoting Effect in Mature and Aged Rats

Synthetic peptide No. 1 was administered to mature female rats (eightmonths old, ten rats in each group) by oral gavage using a gastric tubeonce a day for 20 weeks. The dose of the synthetic peptide was 1, 10 or100 mg/kg·day. As a positive control, alendronate sodium hydrate(hereinafter called “alendronate”) (300 μg/kg) was subcutaneouslyadministered once a day for 20 weeks. The results revealed that the bonedensity in the femurs and the cancellous bone volume in the proximaltibial metaphysis at the end of the administration were significantlyincreased in the synthetic peptide administration group and thealendronate administration group as compared with the control group. Thelevels of the increases in the synthetic peptide administration groupwere comparable to those in the alendronate administration group.

Synthetic peptide No. 1 was administered to aged female rats (13 to 14months old, ten rats in each group) by oral gavage using a gastric tubeonce a day for 20 weeks. The dose of the synthetic peptide was 1, 10 or100 mg/kg·day. As a positive control, the alendronate (300 μg/kg) wassubcutaneously administered once a day for 20 weeks. The resultsrevealed that the bone density in the femurs and the cancellous bonevolume in the proximal tibial metaphysis increased in a dose-dependentmanner in the synthetic peptide administration group.

Example 9 Inhibitory Effect on Bone Resorption in Osteoclast CultureSystem

A crude fraction of osteoclasts was prepared from the long bones andscapulae of rabbits. The crude fraction was cultured on dentine slicesin the presence of the synthetic peptides (0.0001 to 1000 μmol/L) for 48hours, resulting in a reduction in the total area of pits in aconcentration-dependent manner. Isolated osteoclasts were also preparedby collagen treatment of the crude fraction of osteoclasts. The isolatedosteoclasts were pretreated with the synthetic peptides (0.0001 to 1000μmol/L) for 15 minutes and then cultured on dentine slices for 24 hours,resulting in a reduction in the total area of pits in aconcentration-dependent manner.

Example 10 Study of Activity of Synthetic Peptides for Promoting theGrowth of Mesenchymal Stem Cells

Mouse mesenchymal stem cells 10T1/2 (JCRB accession No. JCRB9080) or ST2(Riken BRC ID: RCB0224) were seeded at 5000 cells/well in a 96-wellplate containing DMEM medium supplemented with 10% fetal bovine serum.The synthetic peptides were added to the wells at 0.1 to 100 nmol/mL,and the cells were cultured for 72 hours. Cell Counting Kit-8 (DojindoLaboratories) was added at 10 μL/100 μL medium, and the plate wasincubated at 37° C. for 1 to 4 hours. The absorbance at 450 nm wasmeasured with a microplate reader. The results revealed that theaddition of the synthetic peptides significantly increased the growth of10T1/2 and ST2 cells.

Example 11 Study of Activity of Synthetic Peptides for InducingDifferentiation of Mesenchymal Stem Cells

Mouse mesenchymal stem cells 10T1/2 or ST2 were seeded at 5000cells/well in a 96-well plate containing DMEM medium supplemented with10% fetal bovine serum. The synthetic peptides were added to the wellsat 0.1 to 100 nmol/mL, and the cells were cultured for four days. Themedium was replaced with DMEM medium supplemented withβ-glycerophosphoric acid I (10 mM) and ascorbic acid (50 μg/mL). At thismedium replacement, medium with or without the synthetic peptides wasused for the examination of the effect of their presence. The cells werefurther cultured in the medium for three days (in total, seven days ofculture). The cells were fixed, and stained for alkaline phosphataseactivity using Alkaline Phosphatase Substrate Kit I (VECTOR Red) (VectorLaboratories). The results indicated strong alkaline phosphataseactivity in the cells cultured in the presence of the syntheticpeptides. The cells cultured in the absence of the synthetic peptidesdid not show such activity. That is, it was revealed that the additionof the synthetic peptides efficiently induced differentiation ofundifferentiated mesenchymal stem cells into osteoblast-like cells(osteogenic cells) over a relatively short period of time of four days.

Example 12 Study of Activity of Synthetic Peptides for InducingDifferentiation of Chondrogenic Cells

Mouse EC (embryonic carcinoma)-derived cloned cell line ATDC5 (Riken BRCID: RCB0565) with a potential to differentiate into chondrocytes wascultured in DMEM/F-12 medium supplemented with 5% FBS. The ATDC cellswere seeded at 2×10⁴ cells/well in a 48-well plate. After 24 hours ofculture, the culture supernatant was removed, then DMEM/F-12 mediumsupplemented with 10 ng/mL insulin was added, and the synthetic peptideswere added at 0.1 to 100 nmol/mL. As a positive control, 1 and 5 ng/mLBMP-2 or BMP-4 (R&D) was used. The medium was changed every 3 to 4 days.

In order to investigate whether the synthetic peptides have asynergistic effect with BMP-2 or BMP-4, co-addition of the syntheticpeptides at 0.1 to 100 nmol/mL with BMP-2 or BMP-4 at 1 ng/mL wasperformed. The cells were cultured for 14 days with periodical mediumreplacement. The cells were fixed in 10% formalin neutral buffer, andstained with Alcian-blue solution for 4 hours. After washing off thedye, the plate was dried and photographed.

The results revealed that the mouse chondrogenic cells ATDC5 formedAlcian-blue-positive nodules by addition of the synthetic peptides, andthe number of the nodules increased in a concentration-dependent manner.This fact indicated that the synthetic peptides induced thedifferentiation of the ATDC5 cells into chondrocytes. The synergisticeffect with BMP-2 or BMP-4 was also indicated.

Example 13 Analysis of Bone Formation Factors in Osteoblast-Like Cells

Mouse osteoblast-like cell line MC3T3-E1 Subclone-4 (ATCC No. CRL-2593)was cultured at 5% CO₂ and 95% air at 37° C. for 72 hours in 10%FBS-containing α-MEM medium with the addition of the synthetic peptidesat 2.5, 5, or 10 μg/mL. After the end of culture, the alkalinephosphatase (ALP) activity of the osteoblast-like cells was measured bythe p-nitrophenyl phosphate substrate method. The levels of osteocalcin(OCN) in the culture supernatants were determined by ELISA using anosteocalcin measurement kit (Takara Bio). The results indicated that theALP activity and the OCN level had a tendency to increase depending onthe concentration of the synthetic peptides added. ALP and OCN areconsidered to be the factors reflecting bone formation function, andthus the results suggested that the synthetic peptides promote the boneformation function of osteoblast-like cells via such related factors.

Example 14 Study of Effect of Synthetic Peptides for Promoting BoneFormation in Growth Cartilage and Primary Cancellous Bone

One of the synthetic peptides (No. 1) or water for injection (control)was administered to Crlj:W1 male rats at four weeks old by oral gavageusing a gastric tube once a day for seven days (n=3 in each group). Thedose of the synthetic peptide (No. 1) was 100 mg/kg·day. As a positivecontrol, human growth hormone (hGH) (Norditropin, Standard CommodityClassification No. of Japan: 872412) was subcutaneously administered at500 μg/kg·day once a day for seven days. As fluorescent labels for thesite of bone formation, tetracycline (20 mg/kg) was subcutaneouslyadministered on day 4 after the administration of the peptide orcontrol, and then calcein (10 mg/kg) was subcutaneously administered onday 6 after the administration of the peptide or control. After theadministration of the test sample on day 7, the rats were kept underfasting conditions. Twenty-four hours later, the blood was collected.The rats were then euthanized by bloodletting under anesthesia, and thetibiae were harvested (see FIG. 16). Non-decalcified specimens of theproximal tibiae were prepared and photographed under a fluorescentmicroscope (BX-53, OLYMPUS). The bone morphology was measured using amorphometry system (Histometry RT digitizer, System Supply Co., Ltd.)and an analysis software (CSS-840 cancellous bone morphometry version,System Supply Co., Ltd.). The longitudinal growth rate (Lo.G.R.) in thegrowth cartilage, which is the increase in the height per day, wasdetermined by measuring the distance between the tetracycline label andthe calcein label, and dividing the distance by the administrationinterval between the fluorescent labels (two days).

The results revealed that the hGH administration group and the No. 1administration group showed significant promotion of bone growth ascompared with the control group, and that the bone growth-promotingeffect observed in the No. 1 administration group was significant ascompared with the hGH administration group (FIG. 17). The No. 1administration group also showed a high frequency of incorporation ofthe fluorescent labels into the primary cancellous bone as compared withthe control group and the hGH administration group, suggesting that thesynthetic peptide induced fast formation of the cancellous bone in thegrowth phase (FIG. 18, white region).

The measurement of the serum level of insulin-like growth factor (IGF-1)by ELISA (Mouse/Rat IGF-I Quantikine ELISA Kit (MG100), R&D Systems,Inc.) revealed that the IGF-1 levels in the hGH administration group andthe No. 1 administration group were higher than that in the controlgroup (FIG. 19). The increase in the level of IGF-1, which is related togrowth of the bones and muscles in a growth phase, may contribute to thebone growth-promoting effect.

It is apparent from the above results that oral administration of thepeptides of the present invention promotes longitudinal bone growth andthe formation of the cancellous bone, which is the internal structure ofthe bone.

Example 15 Study of Effect of Synthetic Peptides for Promoting BoneFormation in Growth Cartilage and Secondary Cancellous Bone

One of the synthetic peptides (No. 1) or water for injection (control)was administered to Crlj:W1 male rats at four weeks old by oral gavageusing a gastric tube once a day for 21 days (n=3 in each group). Thedose of the synthetic peptide was 100 mg/kg·day. As a positive control,human growth hormone (hGH) (Norditropin, Standard CommodityClassification No. of Japan: 872412) was subcutaneously administered at500 μg/kg·day once a day for 21 days. As fluorescent labels for the siteof bone formation, tetracycline (20 mg/kg) was subcutaneouslyadministered on day 18 after the administration of the peptide orcontrol, and then calcein (10 mg/kg) was subcutaneously administered onday 20 after the administration of the peptide or control. After theadministration of the test sample on day 21, the rats were kept underfasting conditions.

Twenty-four hours later, the rats were euthanized by bloodletting underanesthesia, and the tibiae were harvested (see FIG. 20). Non-decalcifiedspecimens of the proximal tibiae were prepared and photographed under afluorescent microscope (BX-53, OLYMPUS). The bone morphology wasmeasured using a morphometry system (Histometry RT digitizer, SystemSupply Co., Ltd.) and an analysis software (CSS-840 cancellous bonemorphometry version, System Supply Co., Ltd.). The longitudinal growthrate (Lo.G.R.) in the epiphyseal cartilage, which is the increase in theheight per day, was determined by measuring the distance between thetetracycline label and the calcein label, and dividing the distance bythe administration interval between the fluorescent labels (two days).

The results revealed that the hGH administration group and the No. 1administration group showed significant promotion of bone growth ascompared with the control group, and that the bone growth-promotingeffect observed in the No. 1 administration group was significant ascompared with the hGH administration group (FIG. 21). It was thusindicated that long-term oral administration of the peptides of thepresent invention also promotes longitudinal bone growth.

The analysis of the secondary cancellous bone volume (bone volume/tissuevolume (BV/TV)) by the above analysis system revealed that the No. 1administration group had a higher secondary cancellous bone volume thanthe control group and the hGH administration group (FIG. 22). The No. 1administration group also showed a high percentage of the bone surfaceoccupied by osteoblasts relative to the total bone surface (osteoblastsurface/bone surface (Ob.S/BS)) (FIG. 23) and a low percentage of thebone surface occupied by osteoclasts relative to the total bone surface(osteoclast surface/Bone surface (Oc.S/BS)) (FIG. 24).

It is apparent from the above results that oral administration of thepeptides of the present invention potentially induces skeletalmetabolism in which bone formation is dominant via an increase ofosteoblasts and inhibition of differentiation into osteoclasts.

Example 16 Study of Effect of Synthetic Peptides for Increasing MineralApposition Rate in Secondary Cancellous Bone

One of the synthetic peptides (No. 1) or water for injection (control)was administered to Crlj:W1 male rats at four weeks old by oral gavageusing a gastric tube once a day for seven days (n=10 in each group). Thedose of the synthetic peptide was 1, 10 or 100 mg/kg·day. As a positivecontrol, human growth hormone (hGH) (Norditropin, Standard CommodityClassification No. of Japan: 872412) was subcutaneously administered at500 μg/kg·day once a day for seven days. As fluorescent labels for thesite of bone formation, tetracycline (20 mg/kg) was subcutaneouslyadministered on day 4 after the administration of the peptide orcontrol, and then calcein (10 mg/kg) was subcutaneously administered onday 6 after the administration of the peptide or control. After theadministration of the test sample on day 7, the rats were kept underfasting conditions. Twenty-four hours later, the rats were euthanized bybloodletting under anesthesia, and the tibiae were harvested.Non-decalcified specimens of the proximal tibiae were prepared andphotographed under a fluorescent microscope (BX-53, OLYMPUS). The bonemorphology was measured using a morphometry system (Histometry RTdigitizer, System Supply Co., Ltd.) and an analysis software (CSS-840cancellous bone morphometry version, System Supply Co., Ltd.). Themineral apposition rate (MAR), per day, in the secondary cancellous bonewas determined by measuring the distance between the tetracycline labeland the calcein label in the secondary cancellous bone, and dividing thedistance by the administration interval between the fluorescent labels(two days). The results revealed a significant increase in the mineralapposition rate in the hGH administration group and the No. 1administration group as compared with the control group. The No. 1administration group also showed a significant increase in the mineralapposition rate at a dose of 10 mg/kg·day or more as compared with thehGH administration group (FIG. 26). The No. 1 administration group alsoshowed a high percentage of the bone surface occupied by osteoblastsrelative to the total bone surface (osteoblast surface/bone surface(Ob.S/BS)) (FIG. 27) and a high number of osteoblasts relative to thebone tissue volume (number of osteoblasts/tissue volume (N.Ob/TV)) (FIG.28). The increase in the mineral apposition rate in the secondarycancellous bone in the No. 1 administration group would have resultedfrom the increase in the number of osteoblasts. The bone formation ratecalculated as the annual fractional volume of bone formed relative tothe bone surface (bone formation rate/bone surface (BFR/BS)) resulted inhigh rates in the No. 1 administration group (FIG. 29). The No. 1administration group also showed a significant increase in the boneformation rate at a dose of 10 mg/kg·day or more as compared with thecontrol group.

It is apparent from the above results that oral administration of thepeptides of the present invention promotes mineral apposition of thesecondary cancellous bone and increases the bone formation rate.

The present invention is not limited to each of the embodiments andExamples described above, and various modifications are possible withinthe scope of the claims. Embodiments obtainable by appropriatelycombining the technical means disclosed in different embodiments of thepresent invention are also included in the technical scope of thepresent invention. The contents of the scientific literature and thepatent literature cited herein are hereby incorporated by reference intheir entirety.

1-32. (canceled)
 33. A method for preventing, treating or alleviating a cartilage disorder or a joint disease, the method comprising administering, to a mammal in need thereof, an effective amount of the peptide having 100 amino acid residues or less comprising an amino acid sequence selected from (a) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser- Pro-Tyr-Glu,

(b) an amino acid sequence derived from the amino acid sequence (a) by conservative substitution or deletion of 1 to 3 amino acids, and (c) an amino acid sequence consisting of at least four contiguous amino acids of the amino acid sequence (a) or (b), or a derivative thereof or a salt thereof.
 34. The method according to claim 33, wherein the peptide or a derivative thereof or a salt thereof can have at least one phosphorylated serine.
 35. The method according to claim 33, wherein the peptide or a derivative thereof or a salt thereof is a fragment of lipovitellin-1.
 36. The method according to claim 33, wherein the peptide or a derivative thereof or a salt thereof has one or more activity which is selected from the group consisting of promotion of chondrocyte growth, promotion of hyaluronic acid production, induction of chondrogenic cell differentiation, promotion of mesenchymal stem cell growth, induction of mesenchymal stem cell differentiation and increase of the serum level of insulin-like growth factor (IGF-1) in a mammal and promotion of osteoblast growth, inhibition of bone resorption.
 37. The method according to claim 33, wherein the peptide is administered orally.
 38. The method according to claim 33, wherein the peptide is included in a medicament, a food or drink, a supplement, a food additive or a cosmetic product.
 39. The method according to claim 33, wherein the peptide or a derivative thereof or a salt thereof, which consists of an amino acid sequence selected from (i) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro- Tyr-Glu, (ii) (SEQ ID NO: 2) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu, (iii) (SEQ ID NO: 3) Val-Asn-Pro-Glu-Ser-Glu-Glu, (iv) (SEQ ID NO: 4) Pro-Glu-Ser-Glu-Glu, (v) (SEQ ID NO: 5) Asp-Glu-Ser-Ser-Pro-Tyr-Glu, and (vi) (SEQ ID NO: 6) Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu.


40. The method according to claim 39, wherein the peptide or a salt thereof has one or more activity which is selected from the group consisting of promotion of chondrocyte growth, promotion of hyaluronic acid production, induction of chondrogenic cell differentiation, promotion of mesenchymal stem cell growth, induction of mesenchymal stem cell differentiation and increase of the serum level of insulin-like growth factor (IGF-1) in a mammal, promotion of osteoblast growth and inhibition of bone resorption.
 41. A method for promoting bone formation, the method comprising administering, to a mammal in need thereof, an effective amount of the peptide having 100 amino acid residues or less comprising an amino acid sequence selected from (a) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu (SEQ ID NO:1), (b) an amino acid sequence derived from the amino acid sequence (a) by conservative substitution or deletion of 1 to 3 amino acids, and (c) an amino acid sequence consisting of at least four contiguous amino acids of the amino acid sequence (a) or (b), or a derivative thereof or a salt thereof, wherein the peptide can have at least one phosphorylated serine.
 42. The method according to claim 41, wherein the peptide or a derivative thereof or a salt thereof has one or more activity which is selected from the group consisting of promotion of chondrocyte growth, promotion of hyaluronic acid production, induction of chondrogenic cell differentiation, promotion of mesenchymal stem cell growth, induction of mesenchymal stem cell differentiation and increase of the serum level of insulin-like growth factor (IGF-1) in a mammal, promotion of osteoblast growth, and inhibition of bone resorption.
 43. The method according to claim 41, wherein the peptide is administered orally.
 44. The method according to claim 41, wherein the peptide is included in a medicament, a food or drink, a supplement, a food additive or a cosmetic product.
 45. The method according to claim 41, wherein said peptide consists of an amino acid sequence selected from the group consisting of (i) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro- Tyr-Glu, (ii) (SEQ ID NO: 2) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu, (iii) (SEQ ID NO: 3) Val-Asn-Pro-Glu-Ser-Glu-Glu, (iv) (SEQ ID NO: 4) Pro-Glu-Ser-Glu-Glu, (v) (SEQ ID NO: 5) Asp-Glu-Ser-Ser-Pro-Tyr-Glu, and (vi) (SEQ ID NO: 6) Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu,

wherein the peptide does not have phosphorylated serine.
 46. The method according to claim 45, wherein the peptide or a salt thereof has one or more activity which is selected from the group consisting of promotion of chondrocyte growth, promotion of hyaluronic acid production, induction of chondrogenic cell differentiation, promotion of mesenchymal stem cell growth, induction of mesenchymal stem cell differentiation and increase of the serum level of insulin-like growth factor (IGF-1) in a mammal, promotion of osteoblast growth, inhibition of bone resorption.
 47. A peptide or a derivative thereof or a salt thereof, which consists of an amino acid sequence selected from (i) (SEQ ID NO: 1) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu-Asp-Glu-Ser-Ser-Pro- Tyr-Glu, (ii) (SEQ ID NO: 2) Val-Asn-Pro-Glu-Ser-Glu-Glu-Glu, (iii) (SEQ ID NO: 3) Val-Asn-Pro-Glu-Ser-Glu-Glu, (iv) (SEQ ID NO: 4) Pro-Glu-Ser-Glu-Glu, (v) (SEQ ID NO: 5) Asp-Glu-Ser-Ser-Pro-Tyr-Glu, and (vi) (SEQ ID NO: 6) Glu-Asp-Glu-Ser-Ser-Pro-Tyr-Glu,

wherein the peptide can have at least one phosphorylated serine.
 48. The peptide or a derivative thereof or a salt thereof according to claim 47, wherein the peptide or a derivative thereof or a salt thereof has one or more activity which is selected from the group consisting of promotion of chondrocyte growth, promotion of hyaluronic acid production, induction of chondrogenic cell differentiation, promotion of mesenchymal stem cell growth, induction of mesenchymal stem cell differentiation and increase of the serum level of insulin-like growth factor (IGF-1) in a mammal, promotion of bone formation, promotion of osteoblast growth, and inhibition of bone resorption. 